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TM248 · Revised 12/18
Program for the Perkin-Elmer GeneAmp
®
PCR System 9700 Thermal Cycler
Preheat the thermal cycler to 94°C before placing tubes inside.
Cycling Profile:
94°C for 2 minutes, then,
94°C for 30 seconds
30% Ramp to 58°C, hold 30 seconds
30% Ramp to 70°C, hold 45 seconds
Repeat for
35 cycles
, then:
68°C for 2 minutes
4°C soak
(9600 default mode)
After completion of the thermal cycling protocol, store samples at –20°C.
4.C. Agarose Gel Electrophoresis
1.
For optimal visualization of the amplification products we recommend using a 4% NuSieve
®
3:1 Plus TBE buffer
precast gel. Alternatively, cast a 4% NuSieve
®
3:1 agarose gel in 1X TBE buffer containing 0.5µg/ml ethidium
bromide. For instructions for the preparation of agarose gels, see Section 7.D.
Note:
The running buffer should contain 0.5µg/ml ethidium bromide.
2.
Dilute the molecular weight marker as follows:
Volume
50bp DNA Step Ladder
12µl
Blue/Orange 6X Loading Dye
4µl
Nuclease-Free Water
8µl
3.
Add 2.5µl of the Blue/Orange 6X Loading Dye to each amplification tube, and mix.
4.
Load 5–10µl of each sample and 10µl of the diluted molecular weight markers onto the gel.
5.
Run the gel in 1X TBE buffer containing 0.5µg/ml ethidium bromide at 5V/cm (measured as the distance
between the electrodes) until the bromophenol blue dye front migrates to the bottom of the gel.
6.
Photograph the gel using a UV transilluminator (320nm).
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